IGEM:PKU Beijing/2009/Notebook/AND Gate 1/Input/2009/07/10/Shuke Wu

Gel electrophoresis
Products of PCR
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
voltage and time: 60V 5min; 120V 15min

Up row:
lane 1~3: tetR+RBS1 1~3;
lane 4~6: tetR+RBS2 1~3;
lane 7~9: tetR+RBS3 1~3;
lane 10: marker;
lane 11~12: tetR+RBS4 2~3;
lane 13~15: tetR+RBS5 1~3;
lane 16~18: tetR+RBS6 1~3;
down row:
lane 1~3: lacI+RBS1 1~3;
lane 4~6: lacI+RBS2 1~3;
lane 7~9: lacI+RBS3 1~3;
lane 10: marker;
lane 11~12: lacI+RBS4 2~3;
lane 13~15: lacI+RBS5 1~3;
lane 16~18: lacI+RBS6 1~3;

result
10 clones were successfully constructed: RBS1~5+lacI and tetR;
2 clones were failed: RBS6+lacI and tetR.

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Plasmid mini prep
6 RBS-tetR: RBS1-tetR1; RBS2-tetR3; RBS3-tetR2, 3 (t2, t3); RBS4-tetR2; RBS5-tetR2;
6 RBS-lacI: RBS1-lacI1; RBS2-lacI1; RBS3-lacI1, 2 (L1, L2); RBS4-lacI3; RBS5-lacI2;

Double digest
L1, L2, t2, t3: Spe1 1uL, EcoR1 1uL, plasmid 4uL, Buffer 2uL, water 12uL
37 ℃ 4 hour

Gel electrophoresis
Products of double digest of L1, L2, t2, t3,
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
Loading buffer and DNA dye: 6×
Voltage and time: 60V 5min; 120V 15min
Lane1: t2: insert 700bp;
Lane2: t3: insert 700bp;
Lane3: marker;
Lane5: L1: insert 1.1kb;
Lane6: L2: insert 1.1kb;

DNA Gel purification
I made a big mistake here. I purified the brightest ones, which are vectors. So I do the double digest again.

Double digest (again)
L1, L2, t2, t3: Spe1 1uL, EcoR1 1uL, plasmid 4uL, Buffer 2uL, water 12uL
37 ℃ over night.